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At least 19 recordsLinked to original sources
PubMed2003

Novel ubiquitin fusion proteins: ribosomal protein P1 and actin.

Ubiquitin is a small, highly conserved protein found in all eukaryotic cells. Through its covalent attachment to other proteins, ubiquitin regulates numerous important cellular processes including apoptosis, transcription, and the progression of the cell cycle. Ubiquitin expression is unusual: it is encoded and expressed as multimeric head-to-tail repeats (polyubiquitins) that are post-translationally cleaved into monomers, or fused with ribosomal proteins L40 and S27a. The ubiquitin moiety is removed from these fusion proteins, but is thought to act as a chaperone in ribosome biogenesis prior to cleavage. Here we show that the chlorarachniophyte algae express several novel ubiquitin fusion proteins. An expressed sequence tag (EST) survey revealed ubiquitin fusions with an unidentified open reading frame (ORF), ribosomal protein P1 and, most interestingly, actin. Actin is an essential component of the eukaryotic cytoskeleton and is involved in a variety of cellular processes. In other eukaryotes, actin genes only exist as stand-alone ORFs, but in all chlorarachniophytes examined, actin is always encoded as a ubiquitin fusion protein. The variety of ubiquitin fusion proteins in these organisms raises interesting questions about the evolutionary origins of ubiquitin fusions, as well as their possible biochemical functions in other processes, such as cytoskeletal regulation.

Actins↗
PubMed1975

Cluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits.

The transducing phage lambdarifd18 isolated by Kirschbaum and Konrad [(1973 J. Bacteriol. 116, 517-526] was found to carry structural genes for several 50S ribosomal proteins and 16S and 23S rRNA. It has previously been demonstrated [Kirschbaum & Scaife (1974) Mol. Gen. Genet. 132, 193-201] that this phage carries genes for the DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) subunits beta and beta'. Thus, the region of the E. coli chromosome carried by lambdarifd18 contains a cluster of genes essential for transcription and translation.

Bacterial Proteins↗
PubMed2003

Translational machinery of channel catfish: II. Complementary DNA and expression of the complete set of 47 60S ribosomal proteins.

Ribosomal protein genes have become widely used as markers for phylogenetic studies and comparative genomics, but they have not been available in fish. We have cloned and sequenced a complete set of all 47 60S ribosomal protein cDNAs from channel catfish (Ictalurus punctatus), of which 43 included the complete protein encoding regions. Most ribosomal protein mRNAs in channel catfish are highly similar to their mammalian counterparts. However, L4, L14, and L29 are significantly shorter in channel catfish than in mammals due to deletions in the 3' end of the gene. Two distantly related L5 cDNAs, L5a and L5b, were found in channel catfish. L5a is more similar to L5 in other vertebrates, while L5b showed significant levels of divergence, suggesting independent evolution of the two L5-encoding genes. The 47 ribosomal protein genes are generally highly expressed and together account for 11-14% of overall gene expression, depending on the tissues. Expression levels were highly variable both within a single tissue among different ribosomal protein genes, and among tissues with regard to a single ribosomal protein gene. Strong tissue preference expression was also observed for some ribosomal proteins. This set of ribosomal protein gene sequences represents one of the most complete sets from any single organism and will aid in fish phylogenetic and comparative genomic studies.

Alternative Splicing↗
PubMed1971

Direction of chain elongation in the formation of Escherichia coli ribosomal protein.

Ribosomal proteins were isolated from logarithmically growing Escherichia coli cells given [(14)C]alanine for short periods. Surprisingly, the specific activity of alanine at the NH(2)-terminal end was higher than that of alanine released by carboxypeptidase A digestion of the ribosomal protein. To determine the direction of chain elongation more precisely, Escherichia coli cells were grown with [(3)H]amino acids, and [(14)C]amino acids were then given to the (3)H-labeled cells as a pulse. Radioactive 30S ribosomal proteins were isolated from these cells and subjected to carboxypeptidase digestion. The ratio of (14)C to (3)H of the released amino acids increased as the time for carboxypeptidase digestion progressed. These observations suggest that some of the ribosomal proteins may be synthesized from the carboxyl end to the amino end through a novel mechanism.

Alanine↗
PubMed2000

Expression, purification, crystallization and preliminary X-ray diffraction studies of bacterial and archaeal L4 ribosomal proteins.

Ribosomal protein L4 is implicated in the peptidyltransferase activity of the ribosome and in certain bacteria it regulates the transcription and translation of the 11-gene S10 operon. The genes for the L4 ribosomal proteins from the hyperthermophilic bacterium Thermotoga maritima and the halophilic archaeon Haloarcula marismortui have been PCR amplified from genomic DNA and cloned under the control of a T7 promoter to generate overexpressing Escherichia coli strains. For both proteins, efficient purification procedures were developed to yield material suitable for crystallization trials. Crystals of T. maritima L4 were obtained in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit, diffracting to 1.7 A resolution with synchrotron radiation. Crystals of H. marismortui L4 belonged to the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution with a rotating-anode source, presumably containing three molecules per asymmetric unit. The results demonstrate that for certain halophilic proteins the same purification and crystallization procedures can be employed as for conventional proteins.

Archaeal Proteins↗
PubMed2002

Comparative anatomy of a regulatory ribosomal protein.

Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.

Amino Acid Sequence↗
PubMed1999

Ribosomal protein S6 cDNA from two Aedes mosquitoes encodes a carboxyl-terminal extension that resembles histone H1 proteins.

Ribosomal protein S6 (rpS6) is the major phosphorylated protein on the eukaryotic ribosome. Because electrophoretic evidence suggested that the homolog of rpS6 from the mosquitoes Aedes albopictus and Aedes aegypti was measurably larger than Drosophila rpS6, we have now isolated full-length cDNAs encoding Aedes albopictus and Aedes aegypti rpS6. The mosquito rpS6 cDNAs encoded a 100 amino acid extension at the carboxyl-terminus, relative to rpS6 from humans and Drosophila. This region had homology to cDNAs encoding histone H1 from various species and accounted for the larger size of the mosquito protein on polyacrylamide gels. On Northern blots, the mosquito cDNA hybridized to a single band measuring approximately 1.2 kb.

Aedes↗
PubMed1977

Functional roles of 50-S ribosomal proteins.

Ribosomal proteins previously inactivated by treatment with fluorescein isothiocyanate have been incorporated into 50-S ribosomal subunits during reconstitution from particles disassembled by 2 M LiCl in the presence of an excess of the modified proteins. The reconstituted particles show alterations in some functional activities resulting from the incorporation of the inactive ribosomal proteins added exogenously. Of the fluorescein-isothiocyanate-treated proteins incorporated, L24 and L25 drastically affect all the activities tested and these proteins possibly play a fundamental role in determining the overall structure of the particle. Proteins L16 and L10 are apparently involved both in the GTP hydrolysis dependent on elongation factor G and in peptidyl transferase activity but the modified protein L11 only affects GTPase activity indirectly and interferes with the ribosome assembly process involving proteins L7 and L12. Protein L1 may be involved with peptidyl transferase activity while proteins L7 and L12, in agreement with many reports in the literature, affect the factor-dependent hydrolysis of GTP.

Escherichia coli↗
PubMed2005

The social life of ribosomal proteins.

Ribosomal proteins hold a unique position in biology because their function is so closely tied to the large rRNAs of the ribosomes in all kingdoms of life. Following the determination of the complete crystal structures of both the large and small ribosomal subunits from bacteria, the functional role of the proteins has often been overlooked when focusing on rRNAs as the catalysts of translation. In this review we highlight some of the many known and important functions of ribosomal proteins, both during translation on the ribosome and in a wider context.

Bacterial Proteins↗
PubMed1999

In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein.

Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable.

ATP-Binding Cassette Transporters↗
PubMed1982

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.

Animals↗
PubMed1998

Anti-Sm autoantibodies cross-react with ribosomal protein S10: a common structural unit shared by the small nuclear RNP proteins and the ribosomal protein.

OBJECTIVE: Cross-reactivity of anti-Sm autoantibodies with a certain ribosomal protein has been reported previously. The present study was undertaken to identify the anti-Sm-reactive ribosomal protein, and to characterize the cross-reactive epitope. METHODS: Two-dimensional gel electrophoresis followed by immunoblotting was used to identify the ribosomal protein (S10) which was reactive with the Y12 anti-Sm monoclonal antibody (MAb). Human anti-Sm antibodies were also tested for cross-reactivity with the Sm-B/B', Sm-D, and isolated S10 proteins by immunoblotting. Epitope analysis was performed by immunoprecipitation of in vitro-translated products of the recombinant S10 and its various mutants. RESULTS: The Y12 MAb and the affinity-purified human anti-Sm autoantibodies cross-reacted with ribosomal S10 protein. Reactivity of the Y12 MAb with S10 protein was abolished by deletion of 19 amino acids at the carboxyl-terminus of S10, containing the Gly-Arg-Gly sequence motif shared by Sm-B/B' and Sm-D (D1 and D3). Replacements of Arg-158 with Gly and of Arg-158/Arg-160 with Gly/Gly at the carboxyl-terminal 157-Gly-Arg-Gly-Arg-Gly region disrupted the Y12 MAb recognition. CONCLUSION: At least a part of human anti-Sm antibodies and Y12 MAb show cross-reactivity among Sm-B/B', Sm-D, and ribosomal protein S10. The carboxyl-terminal Gly-Arg-Gly region of S10 protein is involved in constructing the cross-reactive epitope. This demonstrates that a common structural feature is shared by the ribosomal protein and the small nuclear RNP proteins.

Amino Acid Sequence↗
PubMed1991

Primary structure of rat ribosomal protein S2. A ribosomal protein with arginine-glycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids.

The amino acid sequence of the rat 40 S ribosomal subunit protein S2 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein. Ribosomal protein S2 has 293 amino acids and has a molecular weight of 31,211. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 23-28 copies of the S2 gene. The mRNA for the protein is about 1,000 nucleotides in length. The highly conserved repetitive mammalian gene family designated LLRep3, but not identified before, encodes ribosomal protein S2. Rat S2 is related to Saccharomyces cerevisiae S4, Methanococcus vannielii S5, Escherichia coli S5, and other members of the prokaryotic S5 family. S. cerevisiae S4 and E. coli S5 are involved in the binding of aminoacyl-tRNA to ribosomes and in conditioning the fidelity of translation; it is plausible to assume that rat S2 serves similar functions. The NH2-terminal region of S2 is rich in arginine-glycine repeats including eight that occur in tandem and has two consecutive copies of the motif RGGF; these sequences have been associated with nucleolar localization and binding to RNA.

Amino Acid Sequence↗
PubMed1993

Derivatives of the yeast mitochondrial ribosomal protein MrpS28 replace ribosomal protein S15 as functional components of the Escherichia coli ribosome.

The mitochondrial ribosomal protein MrpS28 is considerably larger than its eubacterial homolog, Escherichia coli ribosomal protein S15 (Eco S15). Relative to a region of homology that spans the entire length of the bacterial protein, mature MrpS28 is extended by 117 and 48 amino acids at its amino and carboxyl termini, respectively. Both the amino-terminal and S15-like domains of MrpS28 are essential for function in yeast mitochondria. Here, we show that these same two domains function in E. coli. The S15-like domain of MrpS28 alone complements a cold-sensitive mutation in E. coli strain KR121 that gives rise to reduced levels of Eco S15. However, complementation by the S15-like domain of MrpS28 is inefficient when compared with Eco S15. Surprisingly, the amino-terminal domain of MrpS28, which is apparently a unique component of the mitochondrial ribosome and is unable by itself to complement the cold-sensitive phenotype, enhances the ability of the S15-like domain to support growth of KR121 cells at nonpermissive temperatures. Together, these data suggest that the amino-terminal domain contributes to the fundamental properties of MrpS28 involved in the assembly and function of both mitochondrial and E. coli ribosomes.

Amino Acid Sequence↗
PubMed1977

Exchange of individual ribosomal proteins between ribosomes as studied by heavy isotope-transfer experiments.

Whether the individual ribosomal proteins undergo exchange between robosomes in vivo during cell growth was examined using heavy isotope transfer methodology. E. coli was grown first in a heavy isotope medium in the presence of [3H] leucine and then transferred to normal medium and allowed to grow for one generation in the presence of [14C] leucine. The "heavy" and "light" ribosomes that were present in such cells were separated by sedimentation and the ribosomal proteins resolved by two-dimensional gel electrophoresis. The individual proteins were burnt in O2 and their contents of [3H] and [14C] labels determined. From the analysis of the data we find that the great majority of the ribosomal proteins of E. coli does not undergo exchange during cell growth. Proteins which were found to exchange to varying levels in different transfer experiments were S1, S2, L7/L12, L9, L10 and L33. All of them except L9 exchanged to the same levels in control experiments in which separately grown heavy and light cells were mixed and processed. These proteins therefore undergo exchange during cell breakage and ribosome isolation. Protein L9 consistently showed appreciably greater exchange in transfer experiments as compared to the controls suggesting that it may exchange in vivo.

Bacterial Proteins↗
PubMed1994

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.

Amino Acid Sequence↗